JPH0610139B2 - Mixtures of fibrinolytic compounds and agents for dissolving blood clots in blood vessels - Google Patents

Abstract

Use of the surface active copolymer having the formula HO(C2H4O)b(C3H6O)a(C2H4O)bH wherein a is an integer such that the hydrophobe represented by (C3H6O) has a molecular weight of 950 to 4000 preferably of 1750 - 3500 and b is an integer such that the hydrophile portion represented by (C2H4O) constitutes 50 - 90% by weight of the compound for the manufacture of an injectable pharmaceutical composition for the protection of tissue damaged by ischemia.

Description

DETAILED DESCRIPTION OF THE INVENTION CROSS REFERENCE TO RELATED APPLICATION This application is filed on May 15, 1986, in Application No. 06-86.
It is a partial continuation application of the application of April 29, 1987, which is a continuation application of No. 3,582.

TECHNICAL FIELD The present invention relates to a fibrinolytic mixture and a drug for thrombolysis in blood vessels, and a drug for removing fibrin from a tumor.

Background of the invention "Fibrinolytic enzyme" means any enzyme capable of cleaving fibrin, including streptokinase, urokinase, tissue plasminogen activator (t-PA) produced from cell culture. Tissue plasminogen activator (rt-PA) produced by recombinant DNA technology,
And tissue plasminogen activator (k-PA) produced by prourokinase, but is not limited thereto. However, prourokinase, which is a plasminogen activator precursor, does not have the activity of cleaving (dissolving) fibrin (fibrin) as it is, and is therefore included in the “fibrinolytic enzyme” of the present invention. I can't. By “isotonic solution” or “isoosmotic solution” is meant a solution that has the same or similar osmotic pressure as blood. The terms clot, fibrin clot, and thrombus are each used interchangeably.

Each year, about 550,000 Americans die of a heart attack. More than that, about 700,000 people are still alive with heart attacks. Even if a person who suffers from a heart attack survives, a part of his / her heart will surely die. Myocardial death is called myocardial infarction, 70
90% is due to coronary thrombosis. A thrombus or clot occludes one of the arteries of the heart, blocking the flow of blood to the muscles around it. This deprives the muscle of oxygen and other nutrients. Until now, this process could not be reversed. High-tech equipment in the intensive care unit has helped the patient to survive a partial death of the patient's heart.

In many other tissues, when the blood supply to these tissues is affected by thrombus or embolus, the same condition as described above occurs. Examples are stroke, deep vein thrombosis, and pulmonary embolism.

Another source of fibrin and fibrin is tumors. There is solid evidence that fibrinogenic proteins collect in solid tumors. The anatomical distribution of fibrin in tumors is
Depends on tumor type. In cancer types, fibrin is deposited within the tumor matrix and around the tumor nest, especially around the tumor and at the tumor host interface. In contrast, fibrin is less prominent in older, more central tumor matrices characterized by hardened collagen deposition. Fibrin is also found among cells of each cancer type. In some cases, interepithelial fibrin deposition is associated with tumor necrosis areas. However, the tumor necrosis area is not always the site of fibrin deposition. Fibrin deposition in flesh species has been less well studied than fibrin in cancer species. In lymphomas, fibrin deposition can be observed between each malignant tumor cell and between adjacent apparently reactive benign lymphoid elements. Fibrin has been reported to be expressed within the area of tumor sclerosis, as in Hodgkin's disease. One study has shown that the pattern and extent of fibrin deposition is characteristic for a given tumor. ("Hemostasis and thrombosis, basic principles and clinical practice" edited by JB Lippincott's RWColman et al., "Abnormality of hemostasis in malignant tumors" No. 1145-1157.
See page. 2.) A lack of uniform supply of blood vessels to the tumor hinders diagnostic and therapeutic treatments. For example, hypoxic tumors are largely unresponsive to the effects of many drugs and irradiation. New drugs such as traditional drugs and monoclonal antibody binders are ineffective unless they are delivered to tumor cells. Fibrin deposits that surround certain tumors impede drug delivery to the tumor. The blood supply to the tumor is also hindered by other factors. Blood vessels in tumors are often small and tortuous. The hydrodynamic resistance of such blood vessels further impedes blood flow to the tumor.

Similar conditions occur for various causes during the acute onset of sickle cell anemia. Sickle cells partially occlude small blood vessels, causing localized hypoxia and oxygen. Therefore, the red blood cells become more sickle. This results in a vicious circle called "acute onset". This treatment includes increasing blood flow to the affected area and oxygenation. Other treatments include combinations of calcium channel blockers. Fibrin formation often complicates acute sickle cell onset.

It has been discovered that certain enzymes act on fibrin deposits to open occluded arteries. Enzymes that have been used effectively so far include streptokinase, urokinase, pro-urokinase, tissue plasminogen activator produced by cell culture, and tissue plasminogen activator produced by recombinant DNA technology. and so on. These enzymes are most effective when given shortly after the occlusion of a blood vessel and before irreversibly damaging the heart tissue. In a study of 11,806 patients treated with intravenous or intracoronary streptokinase,
An 18% increase in survival rate was observed. If the treatment was started within 1 hour of the onset of the first pain of a heart attack, the in-hospital mortality rate was reduced by 47%. (February 22, 1986
Day "Lancet" Volume 8478, 397-
(See page 401). Premature lysis of the thrombus has been found to rescue parts of the heart tissue that would otherwise die.
In a study assessing vessel patency using angiography, tissue plasminogen activator completely opened vessels in 61% of 129 patients. For this control, 29% without enzyme treatment. (Ve
"Lancett" by rstraete and others November 2, 1985
Sun, Vol. 8462, pp. 965-969). As the tissue plasminogen activator, it is necessary to add about 100 μl of Tween 80 per 1 liter of the solution to promote the diffusion of the enzyme. ("Thrombus, Hemostasis" by Kominger et al. (Schüttutgart) Volume 46 (2), Volume 561.
Pp. 565 (1981)).

The enzyme used to lyse the thrombus in the blood vessel activates fibrinolysis to effect lysis of the thrombus. Fibrin is a protein produced by the polymerization of fibrinogen. This fibrin forms a gel that holds the thrombus. The fibrin molecules form a clot and are gradually cross-linked to make a more stable clot. The four enzymes, pro-urokinase, urokinase, streptokinase, and tissue plasminogen activator all have similar effects on fibrin. However, they have different toxicities. When the fibrinolytic mechanism is activated near the clot, the clot dissolves. However, when activated systemically throughout the blood circulation, it significantly reduces the body's ability to stop bleeding. Streptokinase and urokinase tend to activate systemic fibrinolysis. Therefore, they were most effective when directly injected into the affected blood vessel. Tissue plasminogen activator, on the contrary,
It is effective only when actually attached to the fibrin. That is, the activity is local to the area of the blood clot itself and does not cause systemic fibrinolysis. Increasing doses, either in an attempt to increase the rate of clot lysis or to dissolve refractory clots, increase the amount of systemic fibrinolysis and the risk of bleeding. This substance can be injected intravenously in the general circulation, circulates harmlessly, contacts the fibrin in the clot, activates it and dissolves it. Tissue plasminogen activator can dissolve highly cross-linked blood clots. That is, it is possible to dissolve blood clots that have been present for several hours. Also, tissue plasminogen activator has less risk of bleeding than other enzymes. Further effective enzyme-based thrombolytic substances have been developed.

Characteristic of new enzyme therapies is that they are extremely complex and not effective for all patients. The clot in the anterior descending branch of the left coronary artery lyses much more easily than the clot in other arteries. If the enzyme is not directly sent to the thrombus by the flow of blood, it has no effect. For various reasons, more blood flows around the clot in the left anterior descending coronary artery than in other aortic thrombi. Moreover, the presence of collateral circulation formed in response to the obstruction of blood flow in the aorta adversely affects recanalization of the occluded aorta. The presence of many collateral vessels is believed to allow blood to bypass the clot, reducing the pressure gradient across the clot. This reduces the flow of blood through the small openings that would survive in the clot, impedes the delivery of the enzyme to the clot and prevents clot lysis.

Even after the blood clot is dissolved, the factors that formed the blood clot remain. This is likely to cause thrombosis and infarction again within hours or days after the clot is lysed. Rethrombosis is one of the three to three successful cases of lysing a blood clot in the initial procedure.
Reported at 0%. Anticoagulants are currently used to prevent the formation of new blood clots, but they tend to cause bleeding. There is a delicate balance between the amount of anticoagulant needed to prevent vascular rethrombosis and the amount that causes significant bleeding.

After irreversible damage, the effect of clot lysis is small. Irreversible disorders are disorders of the myocardium or vascular bed of the tissue that is supplied with blood by its blood vessels. Although this new enzyme therapy is widely practiced, its major problem is to find a way to identify and treat the disease early in the patient's illness and to find a treatment that is as effective as possible after the onset of a blood clot. To find out.

The events controlling flow and dead tissue associated with coronary thrombosis have become better understood in animal studies. The heart has several blood vessels and its muscles receive blood from more than one blood vessel. Therefore, the tissue changes associated with coronary thrombosis are divided into distinct regions. The central region of tissue is almost necrotic. A region of significant ischemia surrounds this. Outside this area is a region of shallow necrosis called the border area. Finally, there is a dangerous area surrounding this entire area. Baboon experiments showed that the central necrotic area was not affected by revascularization after several hours. However, muscle in other areas that were less compromised during the ischemic period could be salvaged. The surprising finding is that lysing the thrombus to produce a complete arteriography was not sufficient to restore normal flow in the majority of animals. (1985, Flamen
g., et al., "J. Clin. Invest," Vol. 75, pp. 84-90).

During the time of occlusion, the area supplied by the blood vessel experienced yet another obstacle to blood flow. In another experiment, it was found that the flow rate through the damaged tissue was high immediately after removing the vascular lesion. However, within a short period of time, the blood flow through the ischemic area diminished and the tissue died. That is, the local blood flow immediately after recanalization is a bad forerunner in rescuing myocardial tissue. If the blood flow through the damaged tissue is maintained at near normal levels, the chances of successful tissue rescue are great. Bleeding occurred only in areas of significant ischemia, which reflected small vessel damage. However, the bleeding was confined to the highly ischemic tissue and did not cause infarct dilation or other significant complications. Treatment that separates the flow of blood through the small blood vessels from the major area of the thrombus after recanalization appears to significantly increase rescue of myocardial tissue.

The cardiomyocyte damage that occurs after lysis of the thrombus is due to ischemia and other factors. When fresh blood comes into contact with damaged or dead cells, it causes an influx of neutrophils or pus cells, killing recoverable heart cells. Many of the disorders caused by neutrophils were due to superoxide ions. Superoxide anions can damage tissue in a variety of ways. The interaction of superoxide anion with hydrogen peroxide produces hydroxyl radicals, which can be toxic and react rapidly with most organic molecules. Mannitol is a selective scavenger of hydroxyl groups. The enzyme or superoxide dismutation enzyme causes the decomposition of the superoxide anion. Enzymes such as superoxide dismutation enzymes, free radical scavengers, or agents that block influx on neutrophils can improve cardiomyocyte rescue.

Lower concentrations of copolymer have less effect on plasma proteins. When the concentration is higher than the critical micelle concentration, complement is activated via the alternative passage. This provides yet another benefit in treating heart attacks. This is because tissue activation of complement renders neutrophils unresponsive to complement chemotaxis,
Prevents their migration to the heart tissue.

Continuous therapy is required after blood flow is restored and damaged tissue is rescued. The arteriosclerosis that caused the original heart attack remains. Researchers in the US and Europe have found that arteriosclerosis narrows the arteries in 70-80% of patients who have clots lysed by thrombolytic therapy. Many doctors
We believe that this obstacle must be opened to the benefit of the long run. Balloon revascularization is a method of inserting a catheter with a small balloon into a narrowed artery.
The balloon is inflated, forcing atherosclerosis against the vessel wall and inflating the artery. The effectiveness of this method is limited by ischemia generated by the balloon, embolization of atherogenic substances present in distal blood vessels, and propensity for immediate or delayed thrombosis in the area damaged by the balloon. It This balloon cuts the tissue that is exposed to the underlying collagen and lipid substances that cause thrombus formation. This thrombus creates a series of events that immediately occlude the blood vessel or cause occlusion after days or weeks. What is needed is to reduce the surface of dilated blood vessels to become a thrombogenic source, improve blood flow through the terminal tissues, and break up occluded material into smaller pieces so that they do not cause obstruction. It is to be.

The lipid material on the walls of atherosclerosis creates a mass of plaques. This plaque narrows the lumen of the artery, creating a prone surface for thrombosis. What is needed is a method to extract or coat lipids from atherosclerotic plaques, which keeps their surfaces non-thrombogenic and reduces their mass.

The use of copolymers made by condensation of ethylene oxide and propylene oxide to treat embolisms and thrombi has been previously described (US Pat. No. 3,6,6).
41, 240). However, the effect is limited to newly formed small (preferably micro) thrombi and embolisms, which are mainly constituted by platelets. The use of copolymers of ethylene oxide and propylene oxide has little effect on the blood clots of patients suffering from severe coronary atherosclerosis. The clot formed in these patients is a large and stable clot. A stable blood clot is a blood clot in which fibrin formed from a fibrin source is crosslinked. Fibrin that is undergoing cross-linking is not affected by the presence of the ethylene oxide-propylene oxide copolymer. This copolymer only has an effect on new clots in which the fibrin is not yet newly crosslinked.

Therefore, there is a need for a drug that can reliably dissolve a blood clot, that is, a drug that can reliably dissolve a blood clot, including that it can prevent the re-formation of a second blood clot after the first clot has disappeared. . Ideally, this mixture should minimize as much as possible all the injuries caused by the interruption of the blood supply to the tissue and that the patient should not suffer any injuries due to the re-formation of blood clots. Be done. There is also a need for agents that remove blood clots from tumors and improve the flow through tortuous pathways, thereby allowing therapeutic agents to be delivered to the tumor.

Accordingly, one object of the present invention is to use a standard amount of enzyme in the combination of a fibrinolytic enzyme and a surface active copolymer to enhance the rate of clot lysis or to use a small amount of enzyme. It is possible to formulate so as to maintain the dissolution effect of blood clots while suppressing side effects.

Another object of the present invention is to provide such a mixture that promotes clot lysis by releasing aggregated platelets and by preventing another platelet from clumping into the clot. is there.

Another object of the present invention is to provide such a mixture that can reduce the dose of fibrinolytic enzyme required to lyse the clot, thereby reducing the risk of complications. .

Another object of the present invention is to provide a drug for dissolving fibrin associated with tumor.

SUMMARY OF THE INVENTION The mixtures provided in accordance with the present invention are effective in lysing blood clots and restoring and maintaining blood flow through occluded coronary arteries and other blood vessels. The fibrinolytic mixture of the present invention consists of enzymes such as streptokinase, urokinase, pro-urokinase, tissue plasminogen activator, or other fibrinolytic enzymes, and surface active copolymers. The surface-active copolymer can be a condensation product of ethylene oxide-propylene oxide and has the general formula:

HO (C ₂ H ₄ O) b (C ₃ H ₆ O) a (C ₂ H ₄ O) bH where a is an integer and the hydrophobic substance represented by (C ₃ H ₆ O) is about 950 ~ 4000 molecular weight, preferably about 1750
˜3500, b is an integer and the hydrophilic moieties represented by (C ₂ H ₄ O) make up about 50-90% by weight of the surface active copolymer. .

The fibrinolytic mixture of this invention is usually administered by intravenous injection to a patient.

The mixture provided by the present invention can be administered to a patient having a blood clot occluding a blood vessel. The combination of a fibrinolytic enzyme according to the invention and a surface-active copolymer increases the flow of blood around the clot and rapidly dissolves the clot. In addition, it protects the patient by preventing the formation of new blood clots and reducing reperfusion injury.

The mixtures provided by the present invention provide a synergistic effect in lysing blood clots and restore and maintain blood flow through occluded coronary arteries and other blood vessels. Furthermore, the fibrinolytic mixture of the present invention is extremely effective in preventing the re-formation of blood clots and in maintaining blood flow through its blood vessels and diseased ischemic tissue.

The fibrinolytic mixture of the present invention enhances the flow of blood through the narrow ducts around the clot and increases the supply of fibrinolytic enzymes to the clot. The present invention also improves the lysis rate of the clot by accelerating the rate of lysis of the clot by the enzyme and accelerating the supply of the enzyme to the clot which is not normally sufficiently exposed to the soluble enzyme. Furthermore, the fibrinolytic mixture of the present invention reduces the required dosage of the enzyme for a particular application, thereby reducing the risk of complications due to side effects due to that enzyme.

The fibrinolytic mixture of the present invention promotes the dissolution of blood clots,
It reduces the risk of immediate rethrombosis by releasing the aggregated platelets and preventing another platelet from aggregating at the clot or clot site. By reducing the risk of immediate rethrombosis, the fibrinolytic mixture of the present invention allows delaying the application of balloon revascularization or other invasive procedures in the treatment of occluded vascular lesions. To do. This delay allows the implementation of the invasive procedure to be selected according to the conditions most suitable for the patient.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the data shown in Table A according to Example I.

Detailed Description of the Disclosure Examples The solutions used to formulate the fibrinolytic mixture of the present invention are as follows, but are not limited thereto. They are salt water (sodium chloride solution containing 1000 cc of purified water containing 8.5 to 9.5 g of sodium chloride), Ringer's solution, lactated Ringer's solution, Krebs Ringer's solution, and various sugar solutions. All these solutions are well known to those skilled in the art. However, the fibrinolytic mixture of the present invention can be administered as a non-isotonic solution.

The surface-active copolymer is preferably an ethylene oxide-propylene oxide condensation product having the general formula:

HO (C ₂ H ₄ O) b (C ₃ H ₆ O) a (C ₂ H ₄ O) bH where a is an integer and the hydrophobic substance represented by (C ₃ H ₆ O) is 950 ~ 4000 molecular weight, preferably about 1750
˜3500, b is an integer and the hydrophilic moieties represented by (C ₂ H ₄ O) make up about 50-90% by weight of the surface-active copolymer. . These copolymers are sold by BASF (Parsippany, NJ) under the generic name Pluronic® Polyol.

The surface active copolymers of the present invention are rapidly cleared from blood metabolized by the human body. The half-life of this copolymer in blood is about 2 hours. The surface-active copolymer in the improved fibrinolytic mixture of the present invention does not covalently bond to any other component in the mixture.

This polymer block is formed by condensing ethylene oxide and propylene oxide at high temperature and high pressure in the presence of a base catalyst. Although the monomer units combine to form the polymer chains in each copolymer, there is some statistical variation in the number of these monomer units. The molecular weights given are an approximation of the average molecular weight of the copolymer in each formulation. The block of propylene oxide and ethylene oxide need not be pure. Small amounts of other substances may be admixed as long as the overall physicochemical properties are not substantially changed. For details of the production of these products, see US Pat. No. 2,674,61.
No. 9, which is incorporated herein by reference.

Examples of ethylene oxide-propylene oxide condensation products that can be used to prepare the fibrin soluble mixtures of the present invention are, but are not limited to, the following copolymers:

1. Polyol containing about 80% by weight of ethylene oxide and having an average molecular weight of 4,700.

2. Polyol containing about 50% by weight of ethylene oxide and having an average molecular weight of 3400.

3. A polyol containing about 70% by weight of ethylene oxide and having an average molecular weight of 7,700.

4. Polyol containing about 80% by weight of ethylene oxide and having an average molecular weight of 14,600.

5. Polyol containing about 70% by weight of ethylene oxide and having an average molecular weight of 12,600.

6. Polyol containing about 90% by weight of ethylene oxide and having an average molecular weight of 9500.

Suitable ethylene oxide-propylene oxide copolymers used in the fibrinolytic mixture of the present invention are:
A copolymer having the formula: HO (C ₂ H ₄ O) b (C ₃ H ₆ O) a (C ₂ H ₄ O) bH with a hydrophobic material (C ₃ H ₆ O) having a molecular weight of about 1750. , The total molecular weight of the surface-active copolymer is about 8400.

The concentration of copolymer in the clot-dissolving mixture of the present invention may vary depending on the total amount of solution required in a particular environment. The total amount of copolymer used in the present invention will also vary depending on the size and type of thrombus or embolus, the particular copolymer used, the particular fibrinolytic enzyme used, and the height and weight of the patient. be able to.

This copolymer can be used over a wide range of concentrations without adverse side effects. This copolymer is rapidly and completely discharged. 90% of the administered copolymer is excreted within 3 hours. Due to its low toxicity and rapid elimination from the human body, this copolymer can be administered over extended periods of time.

The fibrinolytic mixture of the present invention can also be used in admixture with blood by standard methods. However,
These solutions are preferably intravenously injected into the bloodstream as a bolus and / or as a slow infusion. These solutions are generally mixed with blood in such a way as to maintain a substantially steady venous pressure.

It is also within the present invention that the solution of the surface-active copolymer and the fibrinolytic enzyme are administered separately. For example, a solution of a surface-active copolymer and a solution of a fibrinolytic enzyme can be prepared separately and administered simultaneously or sequentially to a patient with coronary thrombotic occlusion. Since the simultaneous or sequential administration of the two components (copolymer and fibrinolytic enzyme) of the fibrinolytic mixture according to the invention has the same effect as administering these components together, it is therefore also possible according to the invention It is included.

Fibrinolytic enzymes that can be used in the fibrinolytic mixture of the present invention include, but are not limited to: Streptokinase (registered trademark of Hoechst-Roussel Strep
tase), Urokinase (Abbo, North Chicago, Illinois)
Trademark of Laboratories, Inc.) and tissue plasminogen activator (Sweden, Umea)
Biopool AB). This tissue plasminogen activator can be obtained from eukaryotic cells such as human melanoma cells, and can also be obtained by genetic engineering techniques such as recombinant DNA. Some fibrinolytic enzymes are poorly soluble in water and need to be emulsified with a surface active copolymer prior to administration to a patient.

Ideally, a bolus of copolymer solution without enzyme is infused prior to administering the present invention. For example, a 3% solution of the copolymer in 5% isotonic dextrose is infused over 2 minutes to bring the blood concentration of the copolymer to about 0.6 mg / m 2. Furthermore, the solution of the copolymer is about 25 mg / Kg body weight
It is preferred to infuse intravenously at a rate of 1 / hr to a blood concentration of the copolymer of about 0.6 mg / m which is maintained for 4 days or more after administration of the fibrinolytic mixture of the invention. This treatment helps prevent the re-formation of blood clots.

Although the present description is primarily concerned with heart disease, the fibrinolytic mixtures of the present invention are equally applicable to thrombus in other parts of the human body, such as the brain, feet, lungs, and gastrointestinal tract.

In the present invention, when dissolving a blood clot in a blood vessel, a step of injecting an effective concentration of the surfactant copolymer solution having the following formula into the human body can be provided.

HO (C ₂ H ₄ O) b (C ₃ H ₆ O) a (C ₂ H ₄ O) bH where a is an integer and the hydrophobic substance represented by (C ₃ H ₆ O) is about 950-4000 molecular weight, preferably about 175
It is selected to have a molecular weight of 0 to 4000, b is an integer and the hydrophilic moieties represented by (C ₂ H ₄ O) are selected to make up about 50 to 90% by weight of the surface active copolymer. . The blood concentration of this surface-active copolymer is about 0.1
6 mg / m, preferably 0.5-2 mg / m
Is. Next, a solution having an effective concentration of the fibrinolytic enzyme and an effective amount of the surface-active copolymer is injected into the human body.
After the clot is dissolved, a solution with an effective concentration of surface-active copolymer is injected into the human body. The concentration of copolymer is maintained at about 0.4-2 mg / blood for about 4-144 hours.

The present invention also includes a method for removing fibrin from a tumor, which comprises the step of injecting a solution containing a clot-dissolving composition into a living body. That is, the drug comprises a fibrinolytic mixture, the mixture consisting of an effective amount of a fibrinolytic enzyme and an effective amount of a surface active copolymer having the formula:

HO (C ₂ H ₄ O) b (C ₃ H ₆ O) a (C ₂ H ₄ O) bH where a is an integer and the hydrophobic substance represented by (C ₃ H ₆ O) is about 950-4000 molecular weight, preferably about 175
It is selected to have a molecular weight of 0 to 4000, b is an integer and the hydrophilic moieties represented by (C ₂ H ₄ O) are selected to make up about 50 to 90% by weight of the surface active copolymer. . The agent for removing fibrin from the tumor of the present invention is
Can be administered directly to the tumor.

Removal of fibrin from the tumor facilitates access of diagnostic reagents to the tumor. This method also enhances tumor accessibility and sensitivity to chemotherapy.

The specific examples below are applications of the invention, in particular for lysing blood clots in blood vessels and preventing re-formation of blood clots. Other examples will be apparent to those skilled in the art, and the invention is not limited to the specific examples below.

Example I Addition of a copolymer to a clot-dissolving enzyme has a synergistic effect on the clot-dissolving activity of that enzyme. This is shown in this example.

500-750 for a sterile 1 ml tuberculin syringe
0.6 m of micron glass beads are packed (Polyscience, Warrington, PA). The end of the syringe is closed with a nitics filter and a one-way plastic stopcock. Fresh frozen platelet-poor citrate plasma is spiked with 15 μCi / m ¹²⁵ I of human fibrinogen (Sigma Chemical Co., St. Louis, Mo.).
This radioactive plasma was diluted 1: 2 with normal saline and added to one volume of calcium (American Dad, Aquada, Puerto Rico).
e company) and 4 volumes of diluted plasma to remineralize with calcium.

The radioactive fibrin source is bound to a glass bead column as follows. An equal volume of radiolabeled remineralized plasma is added to the parallel bead column and flushed between the beads with no excess plasma above the beads. This "bead clot" process and operation is all performed at 37 ° C. The beads / plasma clots are incubated for 30 minutes and then washed with normal saline for 30 minutes. During the last 5 minutes of a saline wash, the flow rate is monitored and columns with unusually fast or slow flow rates are excluded from the experiment. A typical average flow rate is 0.2 m / min.

The copolymer used in this example has the formula:

HO (C ₂ H ₄ O) b (C ₃ H ₆ O) a (C ₂ H ₄ O) bH Here, the hydrophobic substance (C ₃ H ₆ O) has a molecular weight of about 1750. Has a total molecular weight of about 8400. This copolymer is usually made up in saline as a 1% by weight stock solution of the copolymer.

Blood containing t-PA with or without this copolymer is passed through the column as follows. 10m
Heparinized whole blood of 1 is taken out fresh and t-PA
(10 μg in 1 m normal saline, single chain, Sigma Chemical Co., St. Louis, MO). 5.5m
Blood is mixed with 0.5 m normal saline or 0.5 m copolymer stock solution. An aliquot of whole blood or an aliquot of whole blood diluted 1: 3 with normal saline is run on each column. Add 3 m of each blood preparation to the reservoir connected to each column. Until all flow is stopped
Collect fractions every minute. Measure the volume in each test tube and
Radioactivity is counted with an acor Analytic gamma counter (TMAnalytic, Elk Grove Village, IL). The appearance of radioactivity in the collection tube indicates lysis of the clot.

A collection of this data is shown in Table A and FIG. FIG. 1 shows the cumulative ¹²⁶ I fibrin released from the column (counts / min) as a function of time.

* Simulated thrombi containing ¹²⁵ I fibrin are prepared as described in the text. The ability of the test preparation to dissolve fibrin is determined by measuring the elution rate of radioactivity from the column. Since the copolymer is not an enzyme and has no reactive groups, it cannot dissolve cross-linked fibrin. However, it increases the fibrinolytic activity of t-PA in this model. This model is designed to simulate the structure and flow conditions of coronary thrombosis.

As can be seen from Table A and FIG. 1, treatment of the radioactive clot with the surface-active copolymer released only little radioactivity, indicating no clot lysis. Administration of t-PA to the clot resulted in the release of radioactivity, indicating that the clot in the column had dissolved. However, when the surface-active copolymer is added to the solution, the dissolution rate of the clot in the column is significantly increased. Thus, the combination of surfactant copolymer and t-PA dissolved the clot in the column at a significantly faster rate than with t-PA alone.

In other experiments, the model was modified by changing the size of the beads, the concentration of plasma used to make the clot, the dilution of blood or the concentration of enzyme or copolymer. In some cases, no whole blood flowed through the column, but the blood with the copolymer flowed at a flow rate of about 0.05 m / min. T-PA in blood was measured by the release of ¹²⁵ I-labeled
The fibrin of such a clot cannot be dissolved at all. This is because blood never flows through the clot. In these situations, the use of the copolymer with blood and t-PA caused a rapid lysis of the clot.

Reference Example Sigma product No. F4883, fibrinogen of lot No. 42H9323 was added to a final concentration of 20.
The concentration of fibrinogen added to the normal saline was adjusted to 0 mg / 1 m and then added to 500 m.
A fibrinogen-added blood sample (hereinafter referred to as sample blood) was prepared by adding fibrinogen to blood so that the g / dL was obtained.

As the polyoxyethylene-polyoxypropylene copolymer, trade name "Pluronic F6" manufactured by BASF Germany
8 "of 10% (w / v) saline solution was used. The final concentration of "Pluronic F68" in the test blood is 1 mg /
m.

As pro-urokinase, product number 107 and lot number 292 manufactured by American Diagnostica were used. The test article was zymogen (1.07 mg / mL, ie 90,
000 IU / mg urokinase equivalent, or 96,300
IU / m) as a frozen solution. The final prourokinase concentration in the blood sample for the viscosity test is 5.5 IU /
L (0.0055 IU / m).

The viscosity of each sample is measured by the Brookfield Corne / Plate viscometer (Viscom
eter).

As a result of the viscosity measurement, the flow ratio [Flow Ratio (amount / time)] shown in Table 1-A and Table 1-B below was obtained.

Table 1-A below shows “Pluronic F68” for sample blood.
And / or is a value (Flow ratio) measured immediately after the addition of pro-urokinase, and Table 1-B shows the flow ratio after incubating at 37 ° C. for 3 hours.

Sample No. (1) flow ratio 9.3 and sample in Table 1-A above
The flow ratio of NO.2 is 8.3, and the sample NO. In Table 1-B.
The flow ratio 17.2 of (1) and the flow ratio 23.9 of sample No. (2) are within the respective error ranges, and no substantial difference is recognized.

Similarly, the flow rate ratio 2 of sample NO. (3) in Table 1-A above
The flow rate ratio of 0.5 and sample NO. (4) is 17.5, and the flow rate ratio of sample NO. (3) is 27.0 and the flow rate ratio of sample NO. (4) is 23.9 in Table 1-B. Each is in an error range, and there is no substantial difference in their viscosities.

The above results indicate that prourokinase does not have a substantial effect on the viscosity of sample blood, whether it is used alone or in combination with polyoxyethylene polyoxypropylene copolymer. It was confirmed that the compound did not show a cleavage (lytic) action on fibrinogen.

Example II A fibrinolytic mixture was tested in an in vitro rat heart model. The detailed design of this configuration has been described elsewhere. (Paulson et al., "Basic Res. C", 1986.
ardiol ", Vol. 81, pp. 180-187. This is incorporated herein by reference. ) This model measures the recovery of isolated hearts from the 30-90 minute ischemic period. During this ischemic period, nutrient flow is reduced to normal 1% or completely stopped, followed by a 10 minute reperfusion period. Measure three parameters. (1) cardiac output (CO); (2) left ventricular systolic blood pressure (LVSP); and (3) left ventricular systole (dp / d)
t). DJPaul assesses heart recovery and the amount of damage
Son, et al., 1984, "Basic Res. Cardiol" 79th
Vol. 551-561.

In this experiment, the heart was perfused with washed human whole blood without heparin. Blood flow was stopped completely for 30 minutes and then reperfused with washed human whole blood for 10 minutes. This human whole blood was heparin-free, but the additives as shown in Table B were added. The copolymer used in this example has the formula:

HO (C ₂ H ₄ O) b (C ₃ H ₆ O) a (C ₂ H ₄ O) bH Here, the hydrophobic substance (C ₃ H ₆ O) has a molecular weight of about 1750. Has a total molecular weight of about 8400. This copolymer is usually made up in saline as a 1% by weight stock solution of the copolymer.

The results of this experiment are as follows. The final concentration of surface-active copolymer used in this example is 0.68 mg / m. The streptokinase used in this example is available from Sigma Chemical Co., St. Louis, MO. Streptokinase is administered at a concentration of 100 units / heart. The results are shown in Table B.

* Combination of copolymer and streptokinase, (a)
Difference in cardiac output (CO) between whole blood ischemia control, (b) copolymer only, (c) streptokinase only p <0.0
5. Student experiments were used to determine the difference between each instrument. p
Results of <0.05 were considered very large.

As can be seen from Table B, the combination of copolymer and streptokinase protected the heart better than either copolymer alone or streptokinase alone.

Example III To treat a patient weighing approximately 180 pounds with pulmonary embolism, 500 mg of urokinase (Abbokinase, Abbot Laboratories, Inc., North Chicago, IL) 105 m
Reconstitute in sterile water. In this urokinase solution,
90 m of 0. 2 containing 6 grams of ethylene oxide-propylene oxide copolymer having the formula:
Add 9% sodium chloride solution.

HO (C ₂ H ₄ O) b (C ₃ H ₆ O) a (C ₂ H ₄ O) bH Here, the hydrophobic substance (C ₃ H ₆ O) has a molecular weight of about 1750. Has a total molecular weight of about 8400. These urokinases and copolymers mix thoroughly to form a homogeneous solution. The final volume of this solution is 1
It is 95m.

The fibrinolytic mixture of the present invention is administered by a constant infusion pump capable of delivering a total volume of 195m. The starting amount of the fibrinolytic mixture according to the invention is administered at a speed of 90 m / h for 10 minutes. The continuous infusion according to the invention is then carried out at a speed of 15 m / h for 12 hours. Some of the fibrin soluble mixtures of the present invention are
At the end of the infusion pump feed cycle it will remain in the tube so a solution of 0.9% sodium chloride for 15 m
The residual solution is flushed out of the tube by dosing at a rate of / hour.

Example IV 75 mg urokinase (Abbot Laborato, North Chicago, IL) to treat patients with coronary thrombosis
ries Abbokinase) is reconstituted in 15.6 m of sterile water. In this urokinase solution, 1 having the following formula
Add 300 grams of a 5% dextrose solution containing 5 grams of ethylene oxide-propylene oxide copolymer.

HO (C ₂ H ₄ O) b (C ₃ H ₆ O) a (C ₂ H ₄ O) bH Here, the hydrophobic substance (C ₃ H ₆ O) has a molecular weight of about 1750. Has a total molecular weight of about 8400. These urokinases and copolymers mix thoroughly to form a homogeneous solution. This solution is then diluted with 5% dextrose to give a final volume of 500 m.

The solution of the present invention was applied to the occluded artery at a speed of 4 m / min.
Inject over a period of up to hours. Periodic angiography is performed to determine the response to the fibrinolytic mixture solution of the present invention.

Example V 105 mg of urokinase (Abbokinase manufactured by Abbot Laboratories, Inc., North Chicago, IL) is used to treat a patient with pulmonary embolism weighing about 180 pounds.
m reconstitute in sterile water. 90 m of this urokinase solution containing 6.0 grams of ethylene oxide-propylene oxide copolymer having the formula:
Of 0.9% sodium chloride solution is added.

HO (C ₂ H ₄ O) b (C ₃ H ₆ O) a (C ₂ H ₄ O) bH Here, the hydrophobic substance (C ₃ H ₆ O) has a molecular weight of about 1750. Has a total molecular weight of about 8400. These urokinases and copolymers mix thoroughly to form a homogeneous solution. This solution is diluted with 0.9 sodium chloride to give a final volume of 195 m.

The patient is administered 137 m of a 5% isotonic dextrose solution in which a 3% weight / volume ethylene oxide-propylene oxide copolymer is dissolved. This gives a blood concentration of the copolymer of about 0.6 mg.
/ M (blood is 8% of body weight).

Immediately after this, the fibrin-soluble mixture according to the invention is treated with 195
Dosing by constant infusion pump with the capacity to deliver a total volume of m. The starting dose of the present invention is administered at a rate of 90 m / hr for 10 minutes. The continuous infusion according to the invention is then carried out at a speed of 15 m / h for 12 hours. Some of the invention will remain in the tube at the end of the feed cycle of the infusion pump, so a solution of 0.9% sodium chloride containing 3.0% copolymer in 15 m
The residual solution is flushed out of the tube by dosing at a rate of / hour.

After dissolution of the clot, the solution of the copolymer is administered by intravenous infusion at a rate of about 25 mg / kg body weight / hour to maintain the blood concentration of the copolymer at about 0.6 mg / m. The administration of the copolymer solution is continued for 4 days after the administration of the fibrinolytic mixture according to the invention.

Example VI 1000 units of heparin (Sigma Chemical Co., St. Louis, Mo.) 200m of normal (0.9%) sodium chloride solution for ex vivo organ preservation in preparation for transplantation
Reconstituted in and added 1.36 g of the inventive copolymer and resuspended washed human whole blood to make the perfusion medium. This copolymer has the formula:

HO (C ₂ H ₄ O) b (C ₃ H ₆ O) a (C ₂ H ₄ O) bH Here, the hydrophobic substance (C ₃ H ₆ O) has a molecular weight of about 1750. Has a total molecular weight of about 8400.

Hearts excised from anesthetized Sprague-Gaudrey rats were perfused with (a) blood and heparin or (b) blood, heparin and copolymers for 10 minutes after 90 minutes of low-flow ischemia. Cardiac output (CO), left ventricular systolic blood pressure (LVSP), and left ventricular systole (dp /
dt) was determined and recovery relative to normal heart was expressed as a percentage. Recovery of ischemic animal hearts that received blood with heparin was poor with 12% CO, 44% LVSP, and 34% dp / dt. Hearts given blood, heparin and copolymers showed excellent recovery, 90% C
O, 92% LBSP, and 84% dp / dt.
For the heparin and copolymer groups, all three parameters are all in the ischemic control group (heparin only)
Were statistically different compared to (P <0.01). The differences between the means were determined by student experiments.

Example VII The combination of superoxide and a suitable copolymer is superior to superoxide dismutase alone in protecting ischemic myocardium from reperfusion injury related to oxygen groups and other factors. Show through experiment.

General anesthesia (thiopental sodium 25 mg / kg)
The animals are intubated and insufflated with 70% oxygen at a rate of 12 breaths / minute. A sufficient level of anesthesia is maintained by intermittent feeding of pentosar clots as needed. After skin preparation, a left anterior thoracotomy is performed, the pericardium is opened and the heart is exposed. Identify the left anterior descending coronary artery,
Separate and wind 1 cm from the beginning with a snare. The temporary left anterior descending coronary artery occlusion is completed by tightening the snare and continuing it for 90 minutes. During this process, the pulse and blood pressure were monitored by Hewlett-Packard Model 7758B8.
Monitor using a channel recorder. Arterial blood pressure is monitored by inserting an 18-gauge indwelling catheter into the right femoral artery and measured by a Hewlett-Packard crystal transducer. The electrocardiographic output of premyocardial ischemia is also monitored.
Upon reperfusion of the ligated tissue 90 minutes after ischemia, the snare is gradually released to prevent the hyperemic response. Prepare a defibrillator in the room, prepare all necessary cardiotonic agents, and deal with situations such as cardiac fibrillation and circulatory weakness due to left anterior descending coronary artery ligation. The therapeutic agent is infused as follows in connection with reperfusion. Approximately 300 tested by the method of McCord, "J. Biol. Chem, 244, 6049 (1969).
Bovine superoxide dismutase with 0 activity unit / mg is obtained from Sigma Chemical Co., St. Louis, MO. Dissolve this in 100m normal salt water,
Infuse intravenously for 90 minutes from 15 minutes before reperfusion. This simulates the effects that occur during the dissolution of coronary thrombus.

A solution of the copolymer was prepared in saline at 2% weight / volume and administered as a bolus intravenously over a period of 2 minutes to achieve a blood level of 0.6 mg / m, followed by about 25 mg / kg / At a constant flow rate of about 0.
A blood level of 6 mg / m is maintained for the rest of the experiment.

The ethylene oxide-propylene oxide copolymer has the general formula:

HO (C ₂ H ₄ O) b (C ₃ H ₆ O) a (C ₂ H ₄ O) bH Here, the hydrophobic substance (C ₃ H ₆ O) has a molecular weight of about 1750. Has a total molecular weight of about 8400.

The synergistic effect of this combination is demonstrated by comparing the results of both dogs treated with the copolymer and superoxide disproportionation enzyme therapy and dogs treated with either substance or not. Be done.

The drug is infused intravenously using an IVAC 560 infusion pump. The snare is released 15 minutes after the infusion is started and the infusion is continued until the total amount of drug has been administered for each group. The chest is closed in layers. Use the thoracic duct to expel the pneumothorax and remove it when spontaneous breathing returns. Intravenous fluid (lactated Ringer's solution) is administered to compensate for the 24-hour NPO period prior to surgery, and an additional 3 to 1 ratio to compensate for blood loss. These animals are
Maintain for 4 hours and watch carefully. Each animal is then returned to the operating table and the previous incision is reopened under general anesthesia. Animals are sacrificed by overdosing with barbiturates. Excising the heart and 4 cm proximal to the ascending aorta,
At this time, the origin of the coronary artery must be included.

The same procedure is applied to all groups to identify areas of the myocardium at risk of or actually infarcted.

This technique involves perfusing the left anterior descending coronary artery with 2,3,5 triphenyltetrazolium chloride. This stains the intact myocardium red and not the infarcted myocardium. The borders of the myocardial critical area are determined by perfusing the coronary system with the Evans blue stain via the aortic pathway. This danger zone is demarcated by not being colored with Evans blue.

Of course, the above description relates only to the preferred embodiments of the invention, and many changes and modifications are possible without departing from the scope of the invention as claimed.

EFFECTS OF THE INVENTION As described above, according to the fibrinolytic mixture according to the present invention and the clot-dissolving drug in blood vessels, it is possible to re-form the second clot after the first clot disappears. It can surely dissolve or remove fibrin and blood clots, including prevention. In addition, a drug for removing fibrin from a tumor can reliably remove fibrin or blood clots from the tumor.

 ─────────────────────────────────────────────────── ─── Continuation of the front page Pending examination priorities (56) References JP 59-59629 (JP, A) JP 61-176532 (JP, A)

Claims (14)

[Claims] 1. A fibrinolytic enzyme and a surface-active copolymer, said surface-active copolymer having the formula: HO (C ₂ H ₄ O) b (C ₃ H ₆ O) a (C ₂ H ₄ O) bH a is an integer, and the hydrophobic portion represented by (C ₃ H ₆ O) is 95
It is selected to have a molecular weight of 0 to 4000, b is an integer and the hydrophilic portion represented by (C ₂ H ₄ O) is selected to make up 50 to 90% by weight of the surface active copolymer. , A fibrinolytic mixture. 2. The fibrinolytic mixture according to claim 1, wherein the fibrinolytic enzyme is selected from the group consisting of tissue plasminogen activator, streptokinase, prourokinase and urokinase. 3. The fibrin-soluble mixture according to claim 1, further comprising an oxygen radical scavenger. 4. The fibrin-soluble mixture according to claim 3, wherein the oxygen radical scavenger is selected from the group consisting of superoxide disproportionation enzyme and mannitol. 5. The surface active copolymer has the formula: HO (C ₂ H ₄ O) b (C ₃ H ₆ O) a (C ₂ H ₄ O) bH hydrophobic moieties (C ₃ H ₆ The fibrin-soluble mixture according to claim 1, wherein the molecular weight of O) is 1750 and the total molecular weight of the surface-active copolymer is 8400. 6. The fibrinolytic mixture of claim 1, wherein the fibrinolytic enzyme and the surface active copolymer are in solution. 7. A mixture of a solution containing an effective amount of a fibrinolytic enzyme and an effective amount of a surface active copolymer having the formula: HO (C ₂ H ₄ O) b (C ₃ H ₆ O ) a (C ₂ H ₄ O) bH a is an integer, and the hydrophobic moiety represented by (C ₃ H ₆ O) is 95
Molecular weight of 0 to 4000, preferably 1750 to 4000
Is selected to have a molecular weight of
An agent for dissolving blood clots in blood vessels, wherein the hydrophilic portion represented by (C ₂ H ₄ O) is selected so as to constitute 50 to 90% by weight of the surface active copolymer. 8. The clot lysing agent according to claim 7, wherein the fibrinolytic enzyme is selected from the group consisting of tissue plasminogen activator, streptokinase, prourokinase and urokinase. 9. The drug for dissolving blood clots in blood vessels according to claim 7, further comprising an oxygen radical scavenger. 10. The drug for dissolving blood clots in blood vessels according to claim 9, wherein the oxygen radical scavenger is selected from the group consisting of superoxide dismutation enzyme and mannitol. 11. The surface active copolymer has the formula: HO (C ₂ H ₄ O) b (C ₃ H ₆ O) a (C ₂ H ₄ O) bH hydrophobic moieties (C ₃ H ₆ 8. The agent for dissolving blood clots in blood vessels according to claim 7, wherein the molecular weight of O) is 1750 and the total molecular weight of the surface active copolymer is 8400. 12. The drug for dissolving blood clots in blood vessels according to claim 7, wherein the fibrinolytic mixture further comprises an anticoagulant substance. 13. The intravascular clot dissolving agent of claim 7, wherein the fibrinolytic mixture is used in connection with balloon revascularization. 14. A mixture of an effective amount of a fibrinolytic enzyme and an effective amount of a surface active copolymer having the formula: HO (C ₂ H ₄ O) b (C ₃ H ₆ O) a (C ₂ H ₄ O) bH a is an integer, and the hydrophobic moiety represented by (C ₃ H ₆ O) is 95
Molecular weight of 0 to 4000, preferably 1750 to 4000
Has a molecular weight of, b is an integer and (C
An agent for removing fibrin from a tumor, wherein the hydrophilic moiety represented by ₂ H ₄ O) is selected so as to constitute 50 to 90% by weight of the surface active copolymer.